Project 1: Inflammation is widely recognized as an inducer of cancer progression. Inducible nitric oxide synthase (NOS2), cyclooxygenase-2 and cystathionine beta synthase are inflammation markers and are involved in wound healing, angiogenesis, and carcinogenesis. NOS2 up-regulation and increased nitric oxide (NO) production also affects the redox state of cells and induces protein, lipid, and DNA modifications. Recent research by our laboratory led to the novel and clinically significant observation that NOS2 expression is associated with a prognostic basal-like transcription pattern and is an independent predictor of poor survival in women with estrogen receptor (ER)-negative breast tumors. These findings are currently further pursued in collaboration with the laboratory of Dr. David Wink at the NCI. In FY13, this collaboration showed that up-regulation of NOS2 in ER-negative breast cancer cells occurs in response to hypoxia, serum withdrawal, interferon gamma, and exogenous NO, consistent with a feed-forward regulation of NO production by the tumor microenvironment in breast cancer biology. Moreover, we found that key indicators of an aggressive cancer phenotype including increased S100 calcium binding protein A8, interleukin-6 and -8, and tissue inhibitor matrix metalloproteinase-1 are up-regulated by these NOS2-induced stimulants, whereas inhibition of NOS2 in MDA-MB-231 breast cancer cells suppressed the same markers. NO also altered cellular migration and chemoresistance of MDA-MB-231 cells to taxol and other chemotherapeutics. Most notably, MDA-MB-231 tumor xenografts and cell metastases from the fat pad to the brain were significantly suppressed when NOS2 was inhibited in the nude mice. These novel results further link elevated NOS2 to cancer progression and show that NO production regulates chemoresistance and metastasis of breast cancer cells. Having made these observations, we recently, started to evaluate the role of cystathionine beta synthase in breast cancer progression. This enzyme, like NOS2, releases a gaseous signal molecule which is hydrogen sulfide. Hydrogen sulfide like NO stimulates angiogenesis. Preliminary data show that cystathionine, a product of cystathionine beta synthase, accumulates in breast tumors. Project 2: We continued to comprehensively examine the metabolome, proteome and transcriptome of ER-positive and ER-negative breast tumors from African-American and European-American patients for biomarker discovery. The promise of the study is the discovery of novel biomarkers for prognosis, and for elucidating what may drive the aggressiveness of breast cancer in African-American women. Using an untargeted discovery approach and validation of key metabolites, we characterized the metabolomic profile of human breast tumors and uncovered intrinsic metabolite signatures in these tumors. Importantly, the oncometabolite, 2-hydroxyglutarate (2HG), accumulated in a subset of tumors and human breast cancer cell lines. 2HG reached mmolar concentrations comparable to those in isocitrate dehydrogenase (IDH)-mutant gliomas, despite the absence of IDH mutations. Instead, we discovered a significant association between increased 2HG levels and MYC pathway activation in breast cancer, which was corroborated in human mammary epithelial and breast cancer cells with inducible MYC overexpression and knockdown. Further analyses showed a global increase of DNA methylation in 2HG-high tumors and identified a poor survival tumor subtype with distinct DNA methylation, high tissue 2HG, and heightened occurrence in African-American patients. Tumors of this subtype had a stem cell-like transcriptional signature with WNT and MYC pathway activation. These tumors over-expressed glutaminase, suggesting a functional relationship between glutamine and 2HG metabolism in breast cancer. Accordingly, 13C-labeled glutamine was metabolized into 2HG in cells with aberrant 2HG accumulation, whereas pharmacologic and siRNA-mediated inhibition of glutaminase markedly reduced 2HG. Our findings highlight 2HG as a candidate breast cancer oncometabolite associated with MYC activation and poor prognosis. In collaboration with the Sreekumar laboratory at Baylor College of Medicine, we also measured the metabolome of luminal and basal-like breast cancer cell lines using mass spectrometry and linked these metabolites to biochemical pathways using Gene Set Analysis, and developed a novel rank-based method to select pathways on the basis of their enrichment in patient-derived omics data sets and prognostic relevance. Key mediators of the pathway were then characterized for their role in breast cancer progression. We found that pyrimidine metabolism was commonly altered in breast cancer, and one associated key gene, ribonucleotide reductase subunit M2 (RRM2), predicted decreased survival across all breast cancer subtypes, as well as in luminal patients resistant to tamoxifen. Increased RRM2 expression in tamoxifen-resistant patients was verified using tissue microarrays, whereas the metabolic products of RRM2 were higher in tamoxifen-resistant cells and in xenograft tumors. Both genetic and pharmacological inhibition of this key enzyme in tamoxifen-resistant cells significantly decreased proliferation, reduced expression of cell cycle genes, and sensitized the cells to tamoxifen treatment. This study suggests for evaluating RRM2-associated metabolites as noninvasive markers for tamoxifen resistance and its pharmacological inhibition as a novel approach to overcome tamoxifen resistance in breast cancer. Project 3: We started a project evaluating the impact of stressful life events on tumor biology. In a clinical study, we will give breast cancer patients, who are scheduled for breast cancer surgery, a short survey evaluating their perceived stress and social isolation. We will also collect frozen tumor and adjacent normal breast tissue and blood samples from these patients and evaluate whether the breast tissue or the blood samples have a biological signatures related to their perceived stress and social isolation status. We hypothesize that patients with a high perceived stress exposure have a biological signature consistent with a more aggressive disease and poorer survival. The pilot study is designed to collect 100 tumor/normal pairs from consented patients with a completed survey. Currently, we have enrolled 38 patients and have collected fresh-frozen tumors specimens from 25 of those. In this context, we also evaluated the relationship between socioeconomic status and the occurrence of a p53 mutation, a marker of poor outcome, in breast tumors. We analyzed the association of the tumor p53 mutational status with tumor characteristics, education, and self-reported annual household income (HI) among 173 breast cancer patients from the greater Baltimore area, United States. Results: p53 mutational frequency was significantly associated with HI. Patients with $15,000 HI had the highest p53 mutation frequency (21%), followed by the income group between $15,000 and $60,000 (18%), while those above $60,000 HI had the fewest mutations (5%). When dichotomized at $60,000, 26 out of 135 patients in the low income category had acquired a p53 mutation, while only 2 out of 38 with a high income carried a mutation. In the adjusted logistic regression analysis with 3 income categories (trend test), the association between HI and p53 mutational status was independent of tumor characteristics, age, race/ethnicity, tobacco smoking and body mass. Further analyses showed that HI may impact the p53 mutational frequency preferentially in patients who develop an estrogen receptor (ER)-negative disease. Conclusions: HI is associated with the p53 mutational frequency in patients who develop an ER-negative disease.